Methods of treating preeclampsia

ABSTRACT

Methods are provided for the diagnosis and treatment of patients with increased risk of gestational hypertension or preeclampsia. The methods involve measuring serum M-CSF levels, and administration of M-CSF.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 08/212,446, filedMar. 10, 1994.

FIELD OF THE INVENTION

This invention relates to the use of cytokines, particularly M-CSF, forthe diagnosis and treatment of preeclampsia.

BACKGROUND OF THE INVENTION

Preeclampsia is a major cause of maternal and fetal mortality andmorbidity, and is a disease unique to human beings during pregnancy.Recent evidence has indicated that preeclampsia is characterized byplacental maladaptation and body-wide endothelial cell injury. Failureof trophoblastic invasion into myometrial segments of maternal spiralarteries and the production of cytotoxic mediators which cause systemicendothelial damage also seem to be implicated.

Trophoblasts are a unique cell type in that they share characteristicsof both normal and neoplastic tissue. During normal development, likeneoplastic cells, human trophoblasts invade through the extracellularmatrix into the myometrial portion of spiral arteries. However, unlikeneoplastic cells which endlessly invade and finally spread to otherorgans, the invasive properties of the trophoblasts are eventuallybrought under control, further cell differentiation proceeds and cellsenescence occurs. In normal gestation, trophoblasts convert spiralarteries into uteroplacental arteries by the above process. Pijnenborget al., in Trophoblast Research (Denker and Aplin, eds.) Plenum Press,New York, p. 33 (1990). Uteroplacental arteries then dilateapproximately 30-fold as large as the spiral arteries. Resultinghemodynamic changes enable the placental bed to satisfy the increaseddemand for oxygen from the fetus during the latter stages of gestation.In preeclamptic women, however, spiral arteries are not properlyconverted into uteroplacental arteries due to the failure of the secondwave of trophoblastic migration into the myometrium at the beginning ofthe second trimester. Khong et al., Br. J. Obstet. Gynecol.,93:1049-1059 (1986). As a result, preeclamptic women typicallydemonstrate a high-resistance, high-pressure, and low-flow state withintact, non-dilated spiral arteries, Robertson et al., Am. J. Obstet.Gynecol., 155:401-412 (1986), and demonstrate a wide variety of clinicalsyndromes. Therefore, abnormal behavior of the fetus-derived trophoblastappears to be a central aspect of the disease.

Cytokines provide an important communication system in coordinatingimmune and inflammatory responses. Among the cytokines are a number ofcolony stimulating factors (CSFs), which are named for their majortarget cells, including granulocyte (G-CSF), granulocyte-macrophage(GM-CSF) and macrophage (M-CSF, also known as CSF-1) colony stimulatingfactors. Other cytokines include the interleukins, including IL-1through IL-13, which are known to possess varying activities includingbeing involved in hematopoiesis, and providing defenses againstpathogenic infections.

Cytokines are produced in the uterus and placenta during normalpregnancy. Tabibzadeh, Endocrine Reviews, 12:272-290 (1991). In rats, ithas been observed that M-CSF is secreted by uterine gland cells, and thelevels increased approximately one thousand-fold in the first few daysof pregnancy, with the novel receptor present on invasive trophoblastcells. Pollard et al., Nature, 330:484-486 (1987); Uzumaki et al., PNAS,USA, 86:9323-9326 (1989). In humans, the expression and localization ofmRNA for M-CSF have been demonstrated in mesenchymal cells of thechorionic villous stroma, particularly in cytotrophoblasts lining thevillous core and in the cytotrophoblastic core in the first trimester;in villous mesenchymal cells in the second trimester; and in cellslining the villous vessels in the third trimester. Kanazaki et al.,Human Reprod., 7:563-567 (1992); Daiter et al., J. Clin. Endocrinol.Metab. 74:850-858 (1992). Circulating levels of macrophage M-CSF duringpregnancy are also higher than those of non-pregnant women. Yong et al.,Blood, 180:2897-2902 (1992).

Other cytokines have been identified in placenta and/or uterus. TNF-α ispresent in human amniotic fluids and supernatants of placental anddecidual tissues. Jaattela et al., Lab. Invest. 58:48-52 (1988).Trophoblast derived TNFα induces release of human chorionic gonadotropin(hCG) using IL-6 and IL-6 receptor-dependent systems in normal humantrophoblasts. Ying et al., J. Clin. Endocrinol. Metab., 74:184-191(1992). Conversely, hCG and human placental lactogen (hPL) increase theexpression of TNFα. Schafer et al., J. Perinat. Med., 20:233-240 (1992).

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods of detecting,preventing and/or treating preeclampsia by measuring and, if necessary,increasing levels of biological factors which are normally present inthe placenta and/or uterus, but are absent, or present in reducedconcentrations, in preeclamptic women. Preferred biological factorsinclude circulating cytokines, particularly CSFs, such as M-CSF,interleukins, such as IL-11, and growth factors, such as TNF-α. The mostpreferred growth factor for the methods of the present invention isM-CSF.

In one particular embodiment, the present invention comprises a methodof diagnosing the increased risk of gestational hypertension orpreeclampsia comprising measuring serum M-CSF levels in a patientsuffering from, or who may be susceptible to, preeclampsia orgestational hypertension ("GH"). In a preferred embodiment, serum M-CSFlevels are measured in a pregnant woman at about 14 to 16 weeksgestation. These serum M-CSF levels are compared to the serum level ofM-CSF in women with normal pregnancies. As demonstrated later herein,the serum M-CSF levels are correlated with the occurrence ofpreeclampsia or gestational hypertension (GH) at final pregnancyoutcome; hence, the present invention is useful in order to gain anaccurate prediction of increased risk of incidence of preeclampsiaand/or GH at final pregnancy outcome. This information may be used toidentify those women who may benefit from treatment with M-CSF.

The inventor has discovered that serum TNF-α levels are decreased duringthe early stages of preeclamptic pregnancy, and therefore may be ofadditional value in diagnosing and/or treating the increased risk ofgestational hypertension or preeclampsiain early stages. During laterstages of preeclamptic pregancy, TNF-α levels become increased.

In another embodiment, the present invention comprises a method oftreating a woman suffering from, or who may be susceptible to,preeclampsia or GH with M-CSF. In one embodiment, the present inventioncomprises administering therapeutically effective dosages of a cytokine,such as M-CSF, to a patient, usually a pregnant woman who is determinedto be susceptible to, or who suffers from, preeclampsia or GH. Inaddition to M-CSF, the patient may be treated with one or more otherbiological factors, such as IL-11.

In another embodiment, the present invention comprises theadministration of cells which have been treated with, or transfectedwith, a gene encoding for expression of one or more cytokines,particularly CSFs, and more particularly M-CSF. In a preferredembodiment, the cells used are trophoblasts which have been removed fromthe patient, treated with, or transfected with, a gene encoding for thecytokine(s) in order to render the trophoblasts able to express greateramounts of the cytokine(s), and then administered to the patient,usually a pregnant woman who is determined to be susceptible to, or whosuffers from, preeclampsia. Vectors and genes useful for preparing thetransfected cells are described in U.S. Pat. Nos. 4,868,119 and4,879,227, the disclosures of which are hereby incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of the relationship between serum M-CSF levelsmeasured at 16 weeks gestation and final pregnancy outcome (Δ=patientwith GH ("PIH"); ⊙=patient with normal pregnancy).

DETAILED DESCRIPTION OF THE INVENTION

The biological factors useful for the present invention include thosewhich are normally produced in the local environment of proliferatingtrophoblasts. Among the biological factors useful for the presentinvention are cytokines, particularly the colony stimulating factors,such as G-CSF, GM-CSF, and M-CSF and interleukins, such as IL-1 throughIL-13. Other cytokines useful for administration in the methods of thepresent invention include tumor necrosis factor, especially TNF-α,interferons, and growth factors, such as transforming growth factor β,IGF, and FGF. Other biological factors useful for administration in thepresent invention include fatty acid metabolites, such as prostacyclin,leukotrienes and PAF. The preferred biological factors are M-CSF aloneor in combination with GM-CSF, IL-11 or other biological factors.

The present invention also includes methods of determining thebiological factors which are most appropriate for administration to apatient. In determining the appropriate biological factors to beadministered to a patient, the physician may measure the levels ofbiological factors present in the placenta and/or uterus. Those factorswhich are present in reduced levels compared to normal pregnancy may beconsidered as appropriate factors for administration to a patient.

In a particular embodiment of the present invention, pregnant women maybe diagnosed for increased risk of gestation hypertension orpreeclampsia by measuring serum M-CSF levels during the first trimesterof pregnancy [approximately 1 to 14 weeks]. It is preferred that theM-CSF levels be measured at least twice during this period, preferablyincluding a measurement at about 14 to 16 weeks. If the M-CSF levels aresignificantly below normal, a course of treatment via administration ofM-CSF may be used. In general, levels of 500 units/ml M-CSF or above areconsidered normal. However, M-CSF levels of about 500 to about 600units/ml M-CSF may be suspect, and further monitoring, in the form ofassay for TNF-α, or additional measurements of M-CSF, may beappropriate. A unit of M-CSF is equivalent to approximately 12 picogramM-CSF. Thus, units may be measured by direct assay of the amount ofM-CSF present [the exact ng/ml of M-CSF present in serum], or bybioassay [level of activity of the M-CSF in the serum compared to thatof 12 picograms of M-CSF]. If the patient's serum M-CSF level is belowabout 500 units/ml, the patient may be diagnosed as being at increasedrisk of suffering gestational hypertension and/or preeclampsia. A courseof treatment for management of hypertension may be appropriate.

If additional monitoring is called for, such monitoring may beaccomplished by measuring the serum biological activity level of TNF-αin the patient. Serum biological activity levels of approximately 40units/ml or more of TNF-α are generally considered normal. See Keith etal., J. Perinatology, 13:417-418 (1993), and Keith et al. Hypertensionand Pregnancy (1995) 14:81-90, the disclosure of both of these articlesare hereby incorporated by reference. If the serum M-CSF level is belownormal, or suspect, and serum TNF-α levels are also below normal, thepatient may be diagnosed to be at increased risk of sufferinggestational hypertension and preeclampsia.

In another embodiment of the present invention, a patient with increasedrisk of gestational hypertension or preeclampsia is treated viaadministration of M-CSF. Preferably, treatment with M-CSF begins late inthe first trimester, at about 14 to about 16 weeks, or early in thesecond trimester, at about 16 to about 18 weeks, and may continue untilmeasured serum M-CSF levels are elevated to within normal levels.Preferably, treatment with M-CSF is not continued beyond about 30 weeksof gestation. For the purposes of the present invention, normal serumM-CSF levels are defined as those levels typical for pregnant women whodo not suffer from preeclampsia or gestational hypertension, generallyat least approximately 500 units/ml. [500 units/ml≅6 nanogram/ml].Preferably, treatment with M-CSF is discontinued after M-CSF levels arewithin normal levels, and before any adverse effects of M-CSFadministration are observed.

In a preferred embodiment, M-CSF may be administered together with, orprior to, administration of IL-11. Recently, Ireland et al. (Blood84:267a (1994)) reported that interleukin-11 (IL-11) was produced byplacental fibroblasts, and that it appeared to be involved introphoblast differentiation and placentation. In normal pregnancy plasmavolume is typically in an expanded physiologic state, but preeclampsiais a decreased plasma volume state. IL-11 may help increase plasmavolume (Ault et al., Blood 84:276a (1994)) and may modulate productionof TNF-α, which becomes elevated during late pregnancy in cases ofpreeclampsia. Keith et al., (1993), supra, and Keith et al. (1995),supra. Therefore, treatment with M-CSF beginning prior to treatment withIL-11 may prove synergistic. Early treatment with M-CSF [beginning inthe first trimester and continuing approximately until normal M-CSFlevels are achieved] and later treatment with IL-11 [starting in thesecond trimester, preferably at approximately 18 to 22 weeks ofpregnancy and continuing until about 36 weeks of pregnancy] may produceoptimal resolution of preeclampsia.

In the methods of the present invention, M-CSF and/or other factors maybe administered through any known means. Administration should besystemic, e.g., parenteral. The preferred mode of administration issubcutaneous, preferably not more than 1× daily. The therapeuticallyeffective amount should be insufficient to cause a systemic toxicreaction, but sufficient to elicit the desired therapeutic response. Theactual dosing regimen for such formulations will be determined by theattending physician considering various factors which modify the actionof drugs, for example, the condition, body weight, sex and diet of thepatient, time of administration, and the degree of onset of the disease.

In general, a therapeutically effective dose of M-CSF, i.e., an amountsufficient to increase the serum M-CSF level to within normal ranges, isexpected to be in the range of about 1-200 μg/kg/day or more preferablyin the range of about 5-150 μg/kg/day. The methods of the presentinvention may involve a series of administrations of the pharmaceuticalcompositions. Such a series may take place over a period of about 7 toabout 21 days. One or more series may be administered.

In a preferred embodiment wherein IL-11 is administered, atherapeutically effective dose of IL-11 is expected to be in the rangeof about 1-100 μg/kg/day or more preferably in the range of about 1-50μg/kg/day. The IL-11 is preferably administered subcutaneously.

Generally, administration will be initiated at the low end of the dosingrange initially, and the dose will be increased over a preselected timecourse until a positive effect is observed. Subsequently, incrementalincreases in dosage will be made limiting such incremental increases tosuch levels that produce a corresponding increase in effect, whiletaking into account any adverse affects that may appear. The durationand frequency of intravenous or sucutaneous therapy using the method ofthe present invention will vary, depending on the severity of thedisease, or growth factor depletion being treated and the condition andpotential idiosyncratic response of each individual patient. It iscontemplated that the duration of each intravenous application of M-CSFwill be in the range of 12 to 24 hours of continuous administration.Ultimately, the attending physician will decide on the appropriateduration of therapy using the method of the present invention.

For gene therapy, a preferred cell type for administration of thecytokines in the present invention are trophoblasts. As discussed above,trophoblasts are a unique cell type which are active in the uterusand/or placenta during pregnancy.

EXAMPLES EXAMPLE 1 Trophoblast Cell Culture

Placental specimens are obtained from consenting normal pregnant womenand preeclamptic women at the time of delivery. Primary cell cultures oftrophoblasts isolated from human placenta of normal and preeclampticpregnancies are established according to a modified technique of Klimanet al., Endocrinology, 118:1567-1582 (1986), as described herein.

(Day 1) Placenta is obtained and maintained with aseptic technique andrinsed with cold PBS. Several sections (20 grams) of placenta taken fromthe maternal side of the placenta is placed in a sterile petri dish andwashed with cold sterile PBS to rinse off blood clots. Trophoblast cellsare separated from connective tissue materials using a scalpel. Thecells are covered with the petri dish lid, weighed, and added to 50 mlof trypsin solution.

(Day 2) After overnight at 4° C., the cells are mixed with 0.01% DNase I(dry powder) with trypsin, and incubated in water bath at 37° C. for 15minutes. DMEM with fetal calf serum (FCS) 20% 10 ml medium per 50 mlsolution is added and left for 1 minute. The solution is filtered (20μ)and then poured into sterile Gibco flasks. The cells are suspended byshaking the solution. Finally, the cells are centrifuged over acontinuous Percoll gradient.

The trophoblast cells are cultured in DMEM supplemented with 20% FCS onfibronectin-coated plastics, or in endothelial cell growth medium(Gibco) after using vitronectin-containing endothelial cell platingmedium (Gibco) to encourage cell attachment. Since these cells show verylittle proliferative activity, subculturing is not feasible, butfreezing of freshly isolated trophoblastic cells can be performed.Freezing of some of the cells allows multiple experiments on cellsobtained under similar conditions.

JEG-3 choriocarcinoma cell lines may also be used as models of firsttrimester trophoblast effects. JEG-3 choriocarcinoma cell lines arecultured according to standard cell culture techniques, using DMEMsupplemented with 20% FCS in Falcon flasks or Nunc microtiter plates.

After the wells of the Nunc microtiter plates are washed with minimumessential medium, the medium is changed to culture medium supplementedwith M-CSF or appropriate vehicles. The cells are incubated for 24, 48or 72 hrs before the culture media are removed and centrifuged at 600 gfor 10 minutes to eliminate cell debris. The supernatants are stored at-70° C. until assayed for cytokines and fatty acid metabolites. Theplates are fixed with 4% paraformaldehyde in PBS and stained forevaluation of cell morphology and number.

The results of this histological observation are reported in Example 3.

EXAMPLE 2 Measurement of Cytokines and Fatty Acid Metabolites

A. ELISA for Cytokines: Cytokines are measured with commerciallyavailable, enzyme-linked immunosorbent assay kits (ELISA)(GenzymeCorporation, Cambridge, Mass.). The assay is a triple antibody sandwichusing a monoclonal anti-GM-CSF, -G-CSF, -M-CSF, or -TNFα biotinylatedgoat anti-rabbit immunoglobulin with streptavidin-peroxidase, and0-phenylenediamine as the chromogen.

B. Bioassay for TNFα: The L929 mouse fibrosarcoma cell cytotoxicityassay for TNFα bioactivity is performed as follows in 96 well microtiterplates in triplicates. L929 target cells, washed and resuspended inDMEM+10% FCS, are placed at a density of 1.0×10⁵ cells/ml and 250μl/well. After allowing the cells to attach to the well bottomsovernight, the media are removed and replaced with fresh identical mediacontaining test samples. After 16-18 hrs incubation at 37° C., 40 μl ofa 2.5 mg/ml solution of(3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide)(MTT)(SigmaChemical Co., St. Louis, Mo.) in PBS is added to each well. Afterincubation with MTT for two hours at 37° C., the supernatants areremoved and the formazan crystals (reaction products) are solubilizedwith 100 μl of Sorenson's Glycine Buffer. Plates are then read in aspectrophotometer at 550 nm using a microplate reader. FCS controlsshould give an approximate absorbance of 0.650-0.900. Cytotoxicity isexpressed as % of FCS control, subtracted from 1 to yield the cytotoxicindex. The specificity of the assay is confirmed by neutralization ofcytotoxicity with anti-TNFα antibodies.

C. Bioassay for GM-CSF and M-CSF: FDCP-G cells and doubling dilutions ofsamples for assay or recombinant GM-CSF and M-CSF as standards areincubated in triplicates in microtiter plates. After 24 hrspreincubation, [³ H]methyl thymidine is added (0.5 μCi/well). 16-18 hrslater, cells are harvested and radioactive incorporation measured ascounts per min (cpm). To neutralize GM-CSF activity, 160 μl ofneutralizing antibody to GM-CSF is incubated at 4 hrs at 37° C. with 160μl of 5637 CM medium conditioned by incubation with trophoblast tissue,or RPMI/BSA as a control. For recombinant GM-CSF, 160 μl of antibody (10μg/ml) is incubated with 160 μl of 2.5 ng/ml rGM-CSF.

EXAMPLE 3 Evaluation of Trophoblast Cells

A. Cytological Examination: Cells are grown on plastic coverslips withand without added cytokines or anti-cytokine antibodies. At 24, 48 and72 hrs of incubation, cells are fixed with 4% paraformaldehyde in PBS(pH 7.4), stained with toluidine blue, and examined by light microscope,including assessment of the degree of giant cell formation.Photomicroscopy may be performed and evaluated.

B. Proliferation Assays:

1) MTT Assay [Described in Example 2B]: Proliferation expressed as %FCS.

2) [³ H] Thymidine Incorporation: Trophoblast cell lines are washedtwice with PBS and culture medium is reconstituted. The selectedcytokines or antibodies are added to the wells. [³ H] thymidine at aconcentration of 1 μCi per well is added at the same time point as testcompounds, and the cultures are incubated for 24, 48 or 72 hrs.Proliferative response is evaluated from the beta counts of cellsharvested at the end of the incubation period with a cell harvester.

C. Hormone Content Analysis (hCG): The secretion of hCG into the mediaof each culture is identified with a microparticle enzyme immunoassay(Abbott Laboratories, Abbott Park, Ill.) for β-subunit chains. Briefly,diluted samples of conditioned media are treated with anti-hCGantibodies conjugated with alkaline phosphatase. Thisenzyme-antibody-antigen complex is then incubated with anti-hCG-coatedmicroparticles, and an aliquot of this mixture is transferred to a glassfiber matrix. The matrix is then washed to remove unbound material andthe substrate; 4-methylumbelliferyl phosphate is added, and theresulting fluorescence is measured.

D. Double Staining with Ki-67/PKKK1 (Confirmation of Trophoblast CellType): Sterile glass coverslips are placed into the wells of a 24-wellculture plate (Falcon) and precoated with 500 μl of trophoblast mediumor medium containing test compounds. After 24, 48 or 72 hrs of culture,the coverslips are gently washed and fixed with acetone at 4° C. for 5min. The coverslips are then stained immediately as follows: coverslipsare rehydrated in PBS and incubated for 30 min at room temperature withmonoclonal antibodies to PKKK1 (in cytoplasm) and Ki-67 (innucleus)(1/10, Dako) simultaneously. The rest of the staining followsthe procedure described in Chegrin et al., in Growth factors and theovery (Hirshfield ed.) Plenum Press, New York, p.213-220 (1988).

E. Statistical Analysis: Basal levels of cytokines and eicosanoids aretabulated, and the effects of the different test compounds are assessedby comparing changes in the levels of cytokines and eicosanoids by testcompounds. Significance of the differences between mean values of eachexperiment are evaluated by ANOVA. Data are expressed as mean±standarddeviation. A p value<0.05 is considered significant.

F. Results of In Vitro and Ex Vivo Trophoblast Culture Experiments

Since the clinical definition of preeclampsia consists of clinical signsand symptoms in the third trimester of pregnancy, it is impossible toclassify trophoblast cells from first or second trimesters aspreeclamptic. Therefore, culture experiments with M-CSF and GM-CSF wereperformed using either spontaneous abortus speciments in the 1st or 2ndtrimester or using JEG-3 choriocarcinoma cells as a model of 1sttrimester trophoblast cells. In response to 200 U/ml of M-CSF or GM-CSF,proliferation increased (p<0.05) over controls during 72 hours ofincubation (Table 1). In 1st trimester trophoblast cells (n=3spontaneous abortus specimens) proliferation increased in a dosedependent manner (Table 2)

In contrast, third trimester trophoblast cells from normal andpreeclamptic pregnancy displayed no response to M-CSF or GM-CSF (Table3). This would seem consistent with prvious findings in the literaturecited in the background section of the application.

Proliferation Results: Trophoblast cells from normal pregnanciessurvived better than those from preeclamptic pregnancies (Table 1); thedifference was most prominent at day 5 of cell culture.

                  TABLE 1                                                         ______________________________________                                        PROLIFERATION OF JEG-3                                                        CHORIOCARCINOMA CELLS                                                         (MODEL OF 1ST TRIMESTER TROPHOBLAST CELLS)                                                   M-CSF                                                          CONTROL        (200U/ML) GM-CSF(200U/ML)                                      ______________________________________                                        DAY 1  100         124 ± 10.5                                                                           120 ± 9.7                                     DAY 2  145 ± 10.3                                                                             187 ± 15.6                                                                           203 ± 20.1                                    DAY 3  197 ± 25.9                                                                             341 ± 37.1                                                                           337 ± 45.2                                    ______________________________________                                         Data expressed as percent ratio of each value compared to control             (Day 1 without growth factors). % = value (Optical Density) (Day 1)           × 100.                                                             

                  TABLE 2                                                         ______________________________________                                        PROLIFERATION OF 1ST                                                          TRIMESTER TROPHOBLAST CELLS                                                   M-CSF (U/ML)   % CHANGE FROM CONTROL                                          ______________________________________                                        2              1.75 ± 4.45                                                 20             11.6 ± 4.3                                                  200            16.3 ± 5.5                                                  2000           23.1 ± 5.6                                                  ______________________________________                                         (N = 3 Placentae)                                                        

                  TABLE 3A                                                        ______________________________________                                        SURVIVAL OF NORMAL TROPHOBLAST CELLS                                                         M-CSF                                                          CONTROL        (2000 U/ML)                                                                             GM-CSF(2000 U/ML)                                    ______________________________________                                        DAY 1  100         104 ± 13.5                                                                           95 ± 7.2                                      DAY 3   93 ± 10.7                                                                              90 ± 12.3                                                                           96 ± 11.2                                     DAY 5   80 ± 12.8                                                                              82 ± 16.0                                                                           80 ± 12.9                                     ______________________________________                                    

                  TABLE 3B                                                        ______________________________________                                        SURVIVAL OF PREECLAMPTIC TROPHOBLAST CELLS                                                   M-CSF                                                          CONTROL        (2000 U/ML)                                                                             GM-CSF(2000 U/ML)                                    ______________________________________                                        DAY 1  100         97 ± 8.8                                                                             97 ± 9.0                                      DAY 3   84 ± 6.6                                                                              87 ± 11.4                                                                            80 ± 14.4                                     DAY 5   58 ± 13.5                                                                             60 ± 18.7                                                                            66 ± 19.1                                     ______________________________________                                    

G. Results of Placental Bed Biopsy Evaluation of M-CSF.

To evaluate the relationship between M-CSF expression and preeclampticplacental bed lesions, the distribution of M-CSF was studied inplacentalbed biopsies from normal (n=11) and preeclamptic (PE) (n=20)pregnancies. Immunohistochemical staining was performed in formalin orbouin's fixed, paraffin-embedded specimens using monoclonal antibodiesfor recombinant M-CSF, α-actin (a marker for smooth muscle) andmacrophages via a streptavidin peroxidase method. In normal biopsies,the cytotrophoblast and syncytiotrophoblast cells were M-CSF positive.The intramural endovascular trophoblast cells of the uteroplacentalarteries (UPA) had faint M-CSF staining and the vessels had no α-actinstaining. In areas of myointimal proliferation in UPA, M-CSF positivemacrophages and trophoblast cells were present. PE arteries, withatherosis, did not stain for M-CSF but did stain for α-actin.Trophoblast cells were absent from vessel walls, but periarterialcytotrophoblast cells were positive for M-CSF.

EXAMPLE 4 Measurement of Serum M-CSF levels and Correlation WithPreeclampsia and/or Gestational Hypertension ("GH")

The serum levels of M-CSF were measured at 16 weeks gestation in 68women. Twenty two of these patients ultimately developed GH, and 46patients had normal pregnancies. The mean serum M-CSF levels for thepatients later developed GH was significantly lower than the mean forpatients with normal pregnancies. For patients who later developed GH orpreeclampsia, the mean serum M-CSF level was 455.2±10.5 units/mlcompared to a mean serum M-CSF level of 536.1±20.4 units/ml(mean±standard deviation) for patients with normal pregnancy. The pvalue for this measured difference is p<0.01, and is thus considered tobe statistically significant.

Further measurements were taken for the 22 patients who developed GH and21 of the patients with normal pregnancies. For this matched sets ofpatients, M-CSF levels were again lower in patients with GH, (455.2±10.5units/ml v. 530.3±16.5 units/ml, p<0.0001). Serum M-CSF levels wereinversely related to maximum diastolic pressure during pregnancy(R=-0.472, p<0.0005) and maximum systolic pressure during pregnancy(R=-0.472, p<0.0001). Of the 22 patients with GH, 6 were diagnosed aspreeclamptic. None of the patients without GH was diagnosed aspreeclamptic. Four women were diagnosed as suffering from HELLP Syndrome[Hemolysis Elevated Liver enzymes Low Platelets], the most severemanifestation of preeclampsia. All four of these women had serum M-CSFlevels below 500 units/ml. FIG. 1 is a diagram of the relationshipbetween serum M-CSF levels measured at 16 weeks gestation and finalpregnancy outcome (Δ=patient with GH ("PIH"); ⊙=patient with normalpregnancy).

I claim:
 1. A method of treating a patient with increased risk ofgestational hypertension or preeclampsia, said method comprisingadministering an effective amount of M-CSF to raise serum M-CSF tonormal levels.
 2. The method of claim 1, wherein M-CSF is administeredbeginning late in the first trimester or during the second trimester ofpregnancy.
 3. The method of claim 2, where M-CSF is administered duringthe period beginning at approximately 14 to 16 weeks gestation andending at approximately 30 weeks gestation.
 4. The method of claim 1,wherein M-CSF is administered during the period beginning at about 14 to16 weeks gestation, and administration continues until measured serumM-CSF levels are within normal levels.
 5. The method of claim 1, furthercomprising administering a therapeutically effective amount of IL-11. 6.The method of claim 5, wherein M-CSF is administered beginning prior toadministration of IL-11.
 7. The method of claim 6, wherein M-CSF isadministered beginning during the first trimester of pregnancy untilmeasured serum M-CSF levels are within normal levels, and IL-11 isadministered beginning during the second trimester of pregnancy.